CN112143485B

CN112143485B - Nano fluorescent probe for rapidly detecting hydrogen sulfide and preparation method thereof

Info

Publication number: CN112143485B
Application number: CN202011115442.2A
Authority: CN
Inventors: 王鹏; 崔梦园; 任翔宇; 刘天广; 徐艳琪
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-07-21
Filing date: 2020-10-19
Publication date: 2022-03-08
Anticipated expiration: 2040-10-19
Also published as: CN112143485A

Abstract

The invention discloses a nano fluorescent probe for rapidly detecting hydrogen sulfide and a preparation method thereof, wherein the fluorescent probe is PAAO-UCNPs-EM1, and the preparation method comprises the following steps: firstly, preparing UCNPs by adopting a thermal decomposition method, and then preparing a stock solution of a probe PAAO-UCNPs-EM1 in a solution by ultrasonic-assisted self-assembly; the fluorescence converted at PAAO-UCNPs-EM1 is quenched due to Fluorescence Resonance Energy Transfer (FRET) effect, hydrogen sulfide (H)₂S) converting EM1 into EM2, inhibiting FRET effect, and causing fluorescence recovery of PAAO-UCNPs-EM 2; up-conversion luminescence of the probe solution with peak intensities at 540nm, 660nm and 800nm and H₂There is a linear relationship between S concentration, for H₂S detection has lower detection limit and can be used for H in biomedical samples₂High sensitivity and selectivity detection of S.

Description

Nano fluorescent probe for rapidly detecting hydrogen sulfide and preparation method thereof

Technical Field

The invention relates to a fluorescent probe for identifying hydrogen sulfide and a preparation method thereof, belonging to the field of small molecule fluorescent probes.

Background

Hydrogen sulfide (H)₂S) is the third important gas signaling molecule that participates in living systems following nitric oxide and carbon monoxide. It plays a multifunctional role in regulating a wide range of physiological processes, such as neurotransmission, vasodilation, renal excretion, inflammation, cytoprotection of oxidative stress and ischemia reperfusion injury. In mammalian cells, endogenous H₂S production is usually dependent on cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS) catalyzed synthesis. H₂S can also be produced from D-cysteine and catalyzed by D-amino acid oxidase (DAO) and 3-mercaptoacetate-sulfur transferase (3-MST). H in human tissue₂The concentration of S may be in the micromolar range. Used for maintaining physiological cell function. According to record, endogenous H₂Abnormal levels of S are associated with a variety of human diseases, including colorectal cancer, neurodegenerative diseases, alzheimer' S disease and hypertension. At physiological level, H₂S regulates the intracellular redox state and basic signal processing, including the regulation of vascular tone, myocardial contractility, neurotransmission and insulin secretion. Except for H₂In addition to biomarkers for S-related diseases, H has been reported₂S can also inhibit hypochlorous acid-mediated oxidative damage, H₂S-related probes can be used for hypochlorous acid-related studies, therefore, H₂S is and H₂Ideal biomarker for S-related diseases, for H₂S detection is of great significance in the diagnosis of these diseases.

Conventional H₂The S detection method comprises colorimetry, electrochemical analysis and gas detectionAnd (4) performing phase chromatography. Due to complicated sample processing, cell or tissue destruction, it is not suitable to monitor endogenous H in real time and in situ₂And S. However, the appearance of the fluorescent probe is H₂Real-time in situ monitoring of S offers a new option with many advantages, such as high sensitivity and selectivity, low invasiveness and good compatibility.

Compared with the traditional organic fluorescent probe, the up-conversion particles (UCNPs) have multiple characteristics of anti-Stokes shift, high signal-to-noise ratio, high light stability, no flicker, stronger penetrating power, zero autofluorescence background, low cytotoxicity and the like. Because of these advantages, nanoprobes based on UCNPs have attracted a wide range of interest in the biomedical field, particularly in the detection and imaging of living cells and biological analytes in vivo.

Further, electrochromic materials refer to materials having a membrane or membrane system with unique properties, with reversible potential changes and changes in optical properties. Due to their good photoelectric and electrochemical properties and color change corresponding to electric charge, they have been widely used in the fields of liquid crystal color displays, smart windows, automatic dimmers, electronic switches, sensors, LEDs, and corrosion inhibitors. However, few documents describe the application of electrochromic materials to the biological detection and imaging of living systems.

Up-conversion and EM1/PAA based fluorescent nanoprobes and H thereof have not been retrieved up to now₂And the domestic and foreign literature and patent reports of S detection.

Disclosure of Invention

Aiming at the organic small molecule fluorescent probe in the prior art at H₂Various problems faced in the real-time in situ detection of S molecules, it is a first object of the present invention to provide a H having a fast response time and good selectivity₂S nano fluorescent probe; the second purpose of the invention is to provide a preparation method of the nano fluorescent probe.

The invention adopts the following technical scheme to synthesize:

the nano fluorescent probe for rapidly detecting the hydrogen sulfide is characterized in that the nano fluorescent probe is PAAO-UCNPs-EM1, wherein the nano fluorescent probe is mixed with H₂S inverseThe main group is electrochromic material EM1 connected on the probe, and the molecular structural formula is shown as follows:

1,1,4, 4-tetraarylbutadiene (EM1) was found to have significant electrochromic behavior in H₂In the presence of S, it can change from violet to almost colorless diene (EM 2). Its specific absorption wavelength range is between 500-850nm, we use EM1 as H₂Assembly of S-responsive chromophores into UCNPs to develop H-competence based on Foster Resonance Energy Transfer (FRET)₂S-activated fluorescent nanoprobes.

As a preferred technical solution of the present application, the UCNPs are NaYF 4: yb, Tm @ NaYF 4: yb, Er.

In the invention, a novel up-conversion nano probe assembled with electrochromic chromophore is designed for the first time and used for detecting H₂S; the nanoprobe consists of three parts: NaYF₄：Yb，Tm@NaYF₄: yb, Er (UCNPs), 1,1,4, 4-tetraarylbutadiene (EM1) and polyacrylic acid (PAA). Under excitation of 980nm, the nanoprobe can emit green light of 540nm, red light of 660nm and near infrared light of 800 nm. Subsequently, light at 540nm and 660nm can be quenched with EM1 due to the overlapping absorption spectra. At H₂In the presence of S, EM1 can be converted to EM2, resulting in a continuous enhancement of light at 540nm, 660nm and 800 nm. Finally, such nanoprobes can sensitively monitor endogenous and exogenous H in living cells with high selectivity and low cytotoxicity by up-conversion luminescence (540nm and 660nm) imaging₂S。

As a preferred technical solution of the present application, the PAAO, UCNPs, EM1 can be prepared by themselves or purchased commercially.

A nano fluorescent probe for rapidly detecting hydrogen sulfide is disclosed, and the preparation path and the detection method of the nano fluorescent probe are shown in figure 1.

A preparation method of a nano fluorescent probe for rapidly detecting hydrogen sulfide comprises the following steps: firstly, preparing UCNPs by adopting a thermal decomposition method, and then preparing a stock solution of a probe PAAO-UCNPs-EM1 by self-assembly in a solution through ultrasonic assistance

Specifically, the preparation method comprises the following steps: 1mL by volume: 500. mu.L: measuring 300uL of PAAO solution, UCNPs solution and EM1 solution; the UCNPs solution was added to the PAAO solution and distilled water and EM1 solution were added, then the mixture was treated in a sonicator, chloroform was evaporated in the dark, the resulting final solution was centrifuged in a centrifuge, and the collected probe precipitate was dissolved in deionized water.

As a preferred technical scheme of the application, the mass concentration of the UCNPs is 50 mg/mL; the mass concentration of the EM1 is 0.43 mg/mL; the PAAO is prepared from PAA, and the mass concentration of the PAA is 15 mg/mL.

As a preferred technical scheme of the application, the preparation method of the UCNPs comprises the following steps:

(1)NaYF₄: synthesis of Yb, Tm: synthesizing by adopting a high-temperature thermal decomposition method, and firstly preparing thulium chloride, yttrium chloride, erbium chloride and ytterbium chloride; the molar ratio is 75%: 25%: 0.3% 1mmol/L RECl that will contain Y, Yb and Tm₃Adding into a reaction kettle, and then adding 15mL of 1-octadecene and 6mL of oleic acid into the mixture; after evacuation of nitrogen at room temperature for 20 minutes, the solution was heated to 160 ℃ for 30 minutes; the mixture was then cooled to 25 ℃; at the same time, 2.5mmol/L NaOH and 4mmol/L NH are included₄Adding 5mL of methanol solution of F into the solution; after stirring at 25 ℃ for 30 minutes, the mixture was heated to remove methanol; after rapidly raising the temperature of the system to 300 ℃ and continuously heating for 1 hour, stopping heating, and cooling the temperature of the mixture to 25 ℃, then adding absolute ethanol to precipitate the mixture; then the mixture is washed with cyclohexane and absolute ethyl alcohol in a volume ratio of v/v-1/1; after centrifugation, it was dissolved in cyclohexane to obtain NaYF₄: solution UCNP of Yb, Tm;

(2)NaYF₄：Yb，Tm@NaYF₄: synthesis of Yb and Er: using NaYF obtained in step (1)₄: yb and Tm are seeds, NaYF 4: yb, Tm @ NaYF₄: yb, Er, the experimental steps and the square conditions are the same as those of the UCNPs obtained in the step (1).

Preferably, the preparation method of the thulium chloride comprises the following steps: adding 168mg of thulium oxide, 3mL of distilled water and 3mL of hydrochloric acid to a reaction vessel and heating until the powder is completely dissolved; after refluxing for 30 minutes, the mixture was cooled to room temperature; then transferring the solution into a 10mL volumetric flask, and supplementing the solution to 10mL with distilled water to obtain the thulium chloride solution.

Preferably, the yttrium chloride, erbium chloride and ytterbium chloride are prepared as thulium chloride, except that the raw materials are the corresponding oxides.

As a preferred embodiment of the present application, the preparation method of PAAO is as follows: 0.48g NHS and 0.8g EDCI were weighed respectively and added to a solution of 0.6g PAA in 3mL DMF; after stirring for 6 hours, 1mL of DMF containing 233. mu.L of n-octylamine was added to the above mixed solution; after stirring for 12 hours, the solution was purified by dialysis to give a PAAO solution.

As a preferred technical scheme of the application, the preparation method of the EM1 comprises the following steps: to a solution of 101mg EM2 in 1.5mL anhydrous dichloromethane was added 168mg (4-BrC) under argon₆H₄)₃N^·+BF₄ ^-(ii) a The mixture was then stirred at room temperature for 1.5 hours; 20mL of anhydrous ether was added and the precipitate was filtered to give compound EM1 as a pale purple powder.

A detection method of a nano fluorescent probe for rapidly detecting hydrogen sulfide comprises the following steps: adding sodium hydrosulfide aqueous solution into the probe solution, incubating at 37 ℃ for 10min, and measuring different H₂Fitting different peak intensities and H of the up-conversion luminescence spectrum of the probe solution under the S concentration₂The linear relation between S concentrations is used for constructing quantitative detection H₂A fluorescent nanoprobe of S; wherein H₂The linear range of S is 0-10 mu mol/L, and the detection limit is 0.01-0.13 mu mol/L.

Wherein the prepared probe solution is 200. mu.g/mL (1 mL): NaHS solution (100. mu. mol/L, 10. mu.L).

Effective effect

The invention provides endogenous H of an organism based on UCNP, PAAO and EM1₂Compared with the prior art, the nano fluorescent probe for S detection and the preparation method thereof have the advantages ofThe method has the following beneficial effects:

(1) the synthesis of the probe is relatively simple and is compatible with H₂The mechanism of the S response is based on EM1 and H₂Oxidation-reduction reaction between S;

(2) from the color change and the fluorescence intensity change before and after the response, H can be rapidly detected₂S, the purpose of naked eye detection is achieved;

(3) the fluorescent probe is characterized by no fluorescence per se but can react with H₂After S reacts rapidly, strong fluorescence is generated, thereby realizing H₂And S selective rapid detection.

(4) The probe is only connected with H₂S-specific reaction to generate strong fluorescence without reacting with other possible biological solutions, and embodies that the probe is used for H₂S has good selectivity.

Therefore, the invention is simple, quick and high-sensitivity H₂The S molecule specificity detection probe has wide application prospect in the field of biomolecule detection and intracellular imaging.

Drawings

FIG. 1 synthetic route of Probe PAAO-UCNPs-EM1 and its Synthesis method H₂S, a response schematic diagram;

FIG. 2 probes PAAO-UCNPs-EM1 and H₂S, responding to fluorescence emission spectra before and after;

FIG. 3 Probe PAAO-UCNPs-EM1 and varying concentrations of H₂(ii) a fluorescence emission profile after S (0-10. mu. mol/L) incubation;

FIG. 4 fluorescence emission spectra of the probe PAAO-UCNPs-EM1 after reaction with other possible biological solutions;

FIG. 5 intracellular imaging of the probe PAAO-UCNPs-EM1 in different cells.

Detailed Description

The invention will be further elucidated with reference to specific examples. The instruments or reagents used in the following examples are conventional instruments and reagents not described in detail; the test procedures not specifically described are all conventional procedures well known to those of ordinary skill in the art.

Main reagent and instrument

Used in this studyAll chemicals of (a) were purchased from qualified reagent suppliers and used directly. 2- (2-methyl-4H-chromium-4-methylene) malononitrile, 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -benzaldehyde, thulium oxide, yttrium oxide, ytterbium oxide, erbium oxide, 1-octadecene, n-octylamine, oleic acid (i) (2-methyl-4H-chromium-4-methylene)>90％，OA)，NH₄F, NaF, absolute ethanol, NaOH, PAA (MW 3000, 50%), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were obtained from Profenox technologies, Inc. Lipopolysaccharide (LPS), penicillin, streptomycin, and Phosphate Buffered Saline (PBS) were purchased from Shanghai Allantin Biotech Co., Ltd. N, N-Dimethylformamide (DMF), piperidine, cyclohexane, dimethyl sulfoxide (DMSO), chloroform, methanol, NaHS and acetic acid were all purchased from national drug control chemical Co. Dulbecco's Modified Eagle's Medium (DMEM) was obtained from Sammer Feishel technology (China) Co. Human hepatocellular carcinoma (HepG2) cell line, human lung carcinoma (a549) cell line, cervical carcinoma (Hela) cell line and human normal hepatocyte (L02) cell line were purchased from kayaky bio-inc (south beijing, china).

A 980nm laser source (2W fiber output power, shanxi tong photo electronics ltd) was used as an external excitation light source and connected to an F97pro fluorescence spectrophotometer (shanghai prism). Mass spectra were measured by ABI Mariner ESI-TOF spectrometer (Applied Biosystems, Thermo Fisher Scientific, USA). UCL imaging of live cells was recorded using an inverted fluorescence microscope (Leica, DMi8, Germany). The uv-vis spectra data were obtained on a uv-vis spectrophotometer (Onedrop, njin, china).

Example 1

Preparation of fluorescent probes

1. The preparation steps are as follows:

(1) synthesis of metal chloride: thulium oxide (Tm) was added to a 25mL round bottom flask₂O₃168mg), distilled water (3mL) and hydrochloric acid (3mL) and heated until the powder was completely dissolved. After refluxing for 30 min, the mixture was cooled to room temperature. The solution was then transferred to a 10mL volumetric flask and made up to 10mL with distilled water. The thulium chloride solution was used for the next step.

Similarly, solutions of yttrium chloride, erbium chloride and ytterbium chloride were prepared according to the thulium chloride protocol.

(2)NaYF₄：Yb，Tm@NaYF₄: synthesis of Yb and Er: synthesizing by adopting a thermal decomposition method; RECl containing Y, Yb and Tm₃(1mmol/L) (ratio 75%: 25%: 0.3%) was added to a four-necked flask, and then 1-octadecene (15mL) and oleic acid (6mL) were added to the above mixture. After evacuation of nitrogen at room temperature for 20 minutes, the solution was heated to 160 ℃ and held for 30 minutes. The mixture was then cooled to 25 ℃. At the same time, NaOH (2.5mmol/L) and NH will be included₄A solution of F (4mmol/L) in methanol (5mL) was added to the above solution. After stirring at 25 ℃ for 30 minutes, the mixture was heated to 75 ℃ and held for 30 minutes, and the methanol was removed. After rapidly raising the temperature of the system to 300 ℃ and continuously heating for 1 hour, the heating was stopped, and the temperature of the mixture was cooled to 25 ℃, and then a large amount of anhydrous ethanol was added to precipitate the mixture. The mixture was then washed 3 times with cyclohexane and absolute ethanol (v/v-1/1). After centrifugation, it was dissolved in cyclohexane to obtain NaYF₄: yb, Tm.

Similarly, NaYF can be obtained according to the step (2)₄：Yb，Tm@NaYF₄: yb, Er Solutions (UCNPs).

(3) Synthesis of EM 1: diene EM2 may be obtained according to previous synthetic methods. To a solution of EM2(101mg) in anhydrous dichloromethane (1.5mL) under argon was added (4-BrC)₆H₄)₃N·⁺BF₄ ^-(168 mg). The mixture was then stirred at room temperature for 1.5 hours. Anhydrous diethyl ether (20mL) was added and the precipitate was filtered to give Compound EM1(116mg) as a pale purple powder.

(4) Synthesis of PAAO: NHS (0.48g) and EDCI (0.8g) were added to a solution of 0.6g PAA in DMF (3 mL). After stirring for 6 hours, DMF (1mL) containing n-octylamine (233. mu.L) was added to the above mixed solution. After stirring for 12 hours, the solution was purified by dialysis for 3 days. PAAO solution was obtained.

(5) Synthesis of Probe PAAO-UCNPs-EM 1: mu.L of the UCNPs solution obtained in step (2) was added to the PAAO solution obtained in step (4), 2mL of distilled water and the EM1 solution obtained in step (3) (DMSO dissolution) were added, and the mixture was placed in a sonicator for 10 minutes (intensity 20%, sonication 3 seconds, 5 second interval). The sonicated product was stirred in the dark for 2 hours to evaporate the chloroform. The resulting final solution was centrifuged in a centrifuge (13000 rpm/min). And the collected probe precipitate was dissolved in deionized water.

2. Preparation of EM1, EM2, Probe solution and different concentration gradient NaHS solution

We prepared EM1 solution in DMSO; probe solutions were prepared at different concentrations using PBS solution (pH 7.4, 50% DMSO); preparing NaHS solutions with different concentrations by using distilled water; EM2 solution was obtained from EM1 and H₂And S is obtained after reaction.

Example 2

Probes PAAO-UCNPs-EM1 and H₂High resolution mass spectra of S-responded solutions

The preparation steps of the PAAO-UCNPs-EM1 probe solution and the NaHS solution related to the embodiment are the same as the embodiment 1, and other specific steps are as follows: to the probe solution prepared in example 1, NaHS solution (100. mu. mol/L, 10. mu.L) was added and reacted for 30 seconds, and the reacted solution was diluted with distilled water and subjected to mass spectrum peak measurement by ABI Mariner ESI-TOF spectrometer.

As shown in FIG. 2, a peak (684.41224) of EM2 appeared in the solution after the reaction, indicating that the probe solution was reacted with H₂The mechanism of the S response is that EM1 attached to the probe acted on it and generated EM 2.

Example 3

The preparation steps of the PAAO-UCNPs-EM1 probe solution and the NaHS solution related to the embodiment are the same as the embodiment 1, and other specific steps are as follows: after NaHS solutions (0 to 10 μmol/L) of different concentrations were added to the probe solution prepared in example 1 to react for 30 seconds, the reacted solution was irradiated with a 980nm laser (100mw) externally attached to an F97pro fluorescence spectrophotometer, and its fluorescence emission spectrum was obtained.

As shown in FIG. 3, we can see that with H₂The increase in the concentration of S, the increase in the fluorescence emission intensity at 540nm and 660nm of the reacted probe solution, and the calculation revealed that the probe PAAO-U wasCNPs-EM1 vs H₂The detection limit of S is low, the detection range can reach 0.01-0.13 mu mol/L, and the detection effect of the probe is good and the sensitivity is high.

Example 4

The preparation steps of the PAAO-UCNPs-EM1 probe solution and the NaHS solution related to the embodiment are the same as the embodiment 1, and other specific steps are as follows: make NaHS solution (100. mu. mol/L) with distilled water and make other possible biological solutions (Fe)³⁺(100μM)，Na⁺(100μM)，Mg²⁺(100μM)，K⁺(100μM)，ClO^-(1mM)，Gly(1mM)，Cys(1mM)，GSH(5mM)，Vc(1mM)，Hcy(1mM)，BSA(10μg/mL)，DTT(1mM)，SO₂(100μM)，S₂O₃ ^2-(100. mu.M)). After the prepared solutions (10. mu.L) were added to the probe solutions prepared in example 1, respectively, and reacted for 30 seconds, the reacted solutions were irradiated with a 980nm laser (100mw) externally connected to an F97pro fluorescence spectrophotometer, and fluorescence emission spectra thereof were obtained.

As shown in FIG. 4, we can see that only H is added₂The solution after S can react with the probe, the fluorescence emission intensity of the reacted probe solution at 540nm and 660nm is enhanced, other possible biological solutions do not react with the probe, and the fluorescence emission intensity at 540nm and 660nm is unchanged. This result indicates that our nanoprobe PAAO-UCNPs-EM1 is on H₂S has selective specificity.

Example 5

The procedure for preparing the PAAO-UCNPs-EM1 probe solution in this example is the same as that in example 1, and the other specific steps are as follows: HepG2 cells were cultured in DMEM medium and placed in CO supplemented with 10% Fetal Bovine Serum (FBS), penicillin (80units/mL) and streptomycin (80mg/L)₂Incubator (37 ℃, 5% CO)₂) Culturing in medium. And L02 cells, Hela cells and A549 cells were incubated in the same manner. When four cells were incubated overnight adherent, we incubated the probe PAAO-UCNPs-EM1 (200. mu.g/mL) with four different cell lines (L02 cells, Hela cells, HepG2 cells and A549 cells) for 6 hours and observed the relative intensities of green and red UCLs on an inverted fluorescence microscope.

As shown in fig. 5, L02 cells incubated with probe PAAO-UCNPs-EM1 showed very weak UCL emission only in the green and red UCL channels, while the other three cell lines showed strong UCL signals in both channels. The experiment shows that the probe PAAO-UCNPs-EM1 successfully realizes the intracellular H₂Bioimaging of S and H of Probe PAAO-UCNPs-EM1 in tumor cells₂S imaging has higher brightness compared with normal cells, and has good application prospect in the fields of biology and medicine.

Claims

1. A nanometer fluorescent probe for rapidly detecting hydrogen sulfide is characterized in that the fluorescent probe is PAAO-UCNPs-EM 1; the main group reacting with hydrogen sulfide is an electrochromic material EM1 connected on a probe, and the molecular structural formula is as follows:

2. the nano fluorescent probe for rapidly detecting hydrogen sulfide as claimed in claim 1, wherein the UCNPs are NaYF 4: yb, Tm @ NaYF 4: yb, Er.

3. The method for preparing nano fluorescent probe for rapidly detecting hydrogen sulfide as claimed in claim 1 or 2, wherein UCNPs are prepared by thermal decomposition method, and stock solution of the probe PAAO-UCNPs-EM1 is prepared by self-assembly in solution with ultrasonic assistance.

4. The preparation method of the nano fluorescent probe for rapidly detecting hydrogen sulfide as claimed in claim 3, characterized by comprising the following steps: 1mL by volume: 500. mu.L: measuring 300uL of PAAO solution, UCNPs solution and EM1 solution; the UCNPs solution was added to the PAAO solution and distilled water and EM1 solution were added, then the mixture was put in a sonicator for adjuvant treatment, followed by evaporation of chloroform in the dark, the resulting final solution was centrifuged in a centrifuge, and the collected probe precipitate was dissolved in deionized water.

5. The method for preparing nano fluorescent probe for rapid detection of hydrogen sulfide of claim 4, wherein both PAAO and EM1 can be prepared by themselves or purchased commercially.

6. The method for preparing nano fluorescent probe for rapidly detecting hydrogen sulfide as claimed in any one of claims 3 to 5, wherein the UCNPs are prepared by the following steps:

(1)NaYF₄: synthesis of Yb, Tm: synthesizing by adopting a thermal decomposition method, and firstly preparing thulium chloride, yttrium chloride and ytterbium chloride; the molar ratio is 75%: 25%: 0.3% 1mmol/L RECl that will contain Y, Yb and Tm₃Adding into a reaction kettle, and then adding 15mL of 1-octadecene and 6mL of oleic acid into the mixture; after evacuation of nitrogen at room temperature for 20 minutes, the solution was heated to 160 ℃ for 30 minutes; the mixture was then cooled to 25 ℃; simultaneously, 5mL of a methanol solution comprising 2.5mmol/L of NaOH and 4mmol/L of NH 4F was added to the above solution; after stirring at 25 ℃ for 30 minutes, the mixture was heated to remove methanol; after rapidly raising the temperature of the system to 300 ℃ and continuously heating for 1 hour, stopping heating, and cooling the temperature of the mixture to 25 ℃, then adding absolute ethanol to precipitate the mixture; then the mixture is washed with cyclohexane and absolute ethyl alcohol in a volume ratio of v/v-1/1; after centrifugation, it was dissolved in cyclohexane to obtain NaYF 4: solution UCNP of Yb, Tm;

(2)NaYF₄：Yb，Tm@NaYF₄: synthesis of Yb and Er: using NaYF4 of step (1): yb and Tm are seeds, NaYF 4: yb, Tm @ NaYF 4: yb and Er, and the experimental steps and conditions are the same as those in the step (1), so that the UCNPs are obtained.

7. The method for preparing nano fluorescent probe for rapidly detecting hydrogen sulfide as claimed in claim 6, wherein the method for preparing thulium chloride is as follows: adding 168mg of thulium oxide, 3mL of distilled water and 3mL of hydrochloric acid to a reaction vessel and heating until the powder is completely dissolved; after refluxing for 30 minutes, the mixture was cooled to room temperature; then transferring the solution into a 10mL volumetric flask, and supplementing the solution to 10mL with distilled water to obtain a thulium chloride solution; the yttrium chloride, the erbium chloride and the ytterbium chloride are prepared with thulium chloride.

8. The method for preparing nano fluorescent probe for rapidly detecting hydrogen sulfide according to claim 4 or 5, wherein the PAAO is prepared by the following steps: 0.48g NHS and 0.8g EDCI were weighed respectively and added to a solution of 0.6g PAA in 3mL DMF; after stirring for 6 hours, 1mL of DMF containing 233. mu.L of n-octylamine was added to the above mixed solution; after stirring for 12 hours, the solution was purified by dialysis to give a PAAO solution.

9. The method for preparing nano fluorescent probe for rapidly detecting hydrogen sulfide as claimed in claim 4 or 5, wherein the EM1 is prepared by the following steps: to a solution of 101mg EM2 in 1.5mL anhydrous dichloromethane was added 168mg (4-BrC) under argon₆H₄)₃N^·+BF₄ ^-(ii) a The mixture was then stirred at room temperature for 1.5 hours; 20mL of anhydrous ether was added and the precipitate was filtered to give compound EM1 as a pale purple powder, wherein EM2 has the following molecular formula:

10. the method for rapidly detecting the nano fluorescent probe for hydrogen sulfide as claimed in claim 1, which is characterized in that: adding sodium hydrosulfide aqueous solution into the probe solution, incubating at 37 ℃ for 10min, and measuring different H₂Fitting different peak intensities and H of the up-conversion luminescence spectrum of the probe solution under the S concentration₂The linear relation between S concentrations is used for constructing quantitative detection H₂A fluorescent nanoprobe of S; wherein H₂The linear range of S is 0-10 mu mol/L, and the detection limit is 0.01-0.13 mu mol/L.